This research is intended to elucidate at the molecular level the mechanisms of spontaneous mutagenesis and associated DNA repair systems and how they are regulated in the bacterium Escherichia coli. Our approach is genetic using the numerous mutant E. coli strains defective in a particular mutagenic or DNA repair pathway. Recent breakthroughs involving the discovery of human genes that confer an increased risk of getting various cancers are a direct consequence of the information gathered over the past 30 years on bacterial mutagenic and repair pathways. These so-called "cancer genes" were characterized in families prone to various cancers and some have been shown to cause defects in mismatch repair and DNA replication fidelity which lead to increased mutation frequencies in other genes causing cancers. We hope to explore the relationship between mutagenesis and fidelity of DNA replication using mutator strains that are defective in some aspect of accurate DNA replication. We recently isolated three new mutator alleles at the gyrA locus, the structural gene for DNA gyrase A subunit. This is the first indication that DNA gyrase is involved in DNA replication fidelity. We wish to characterize this involvement by finding out whether DNA gyrase affects replication accuracy indirectly by altering the degree of supercoiling or directly affects the DNA polymerase at the replication fork. We also plan to determine the mutational spectrum of the gyraA mutator alleles. It is widely believed that oxidative damage caused by reactive oxygen species generated by incomplete reduction of oxygen during respiration is responsible for much of spontaneous mutagenesis that leads to genetic diseases and cancers. We will measure mutation frequencies in an anaerobic chamber for several E. coli mutator alleles that are known to be defective in some aspect of DNA replication fidelity. By comparing these values with aerobic ones, an estimation of how much oxidative damage contributes to the mutator activity can be made. We are using antimutator alleles, which decrease spontaneous mutation frequencies, to show that oxidative damage is an important source of mutagenesis in wild-type cells. Two such E. coli antimutator alleles, recA56 and umuC122, decrease aerobic mutation frequencies but show no differences compared to wild-type values measured in the anaerobic chamber. We plan to characterize this oxygen-dependent mutagenesis pathway in wild-type cells.